N,N-diethylhydroxylamine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June to 15 October, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to OECD guideline 202 and GLP requirements. Concentrations of the test item N,N DIETHYLHYDROXYLAMINE were strongly decreased during the exposition. Indeed, DEHA is known to be oxidized quickly in water by dissolved oxygen. Thus EC50 values determined at 24 and 48h post exposure based on initial measured concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentrations of N,N DIETHYLHYDROXYLAMINE were measured in the test solutions at the beginning and at the end of the test.
After being diluted in acetonitrile and Iso C2 medium, samples are analyzed using high pressure liquid chromatography coupled with mass spectrometry (HPLC/MS).

Vehicle:

no

Details on test solutions:

A stock solution at 100 mg/L was prepared for both preliminary and definitive tests. In the preliminary test, 117 µL of N,N DIETHYLHYDROXYLAMINE were added to 1015 g of dilution water, under stirring during 5 min. The solution was clear and homogeneous. In the definitive test, 234 µL of the test item were added to 2030 g of dilution water under stirring during 5 min. The solution was clear and homogeneous.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus (Cladocera, Crustacea), clone 5 or clone A. Animals come from a stock breeding in the laboratory reared in VOLVIC® water added with vitamin B12, Na2SeO3, 5H2O, calcium and magnesium salts and fed with unicellular green fresh-water algae Pseudokirchneriella subcapitata and Chlorella Vulgaris. Selection of test organisms less than 24 hours-old is made by filtration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no data

Test temperature:

between 19.8 and 20.6 °C

pH:

see details in the Results section

Dissolved oxygen:

see details in the Results section

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations tested in the preliminary test: 100, 50, 10, 5, 1 mg/L
Nominal concentrations tested in the definitive test: 51.4, 23.4, 10.6, 4.8, 2.2, 1 mg/L

Details on test conditions:

Daphnids are exposed to a geometric series of N,N DIETHYLHYDROXYLAMINE test concentrations and a negative control (dilution water) for 48 hours. Four replicate test chambers are maintained in each treatment and control group. The test item concentrations causing 0 and 100 % of immobilisation are determined, and EC50-24 h and EC50-48 h are estimated using appropriate statistical methods.

The study was performed in a temperature-controlled room using 30 mL glass bottles stoppered with PTFE bungs and sealed with aluminium caps. The temperature, continuously recorded in one test flask, remained between 19.8 and 20.6 °C and was constant within ± 1°C according to OECD 202 Guideline. Physical-chemical parameters were measured using a METTLER TOLEDO 345 pH-meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement. Ultra-pure water was used for the preparation of dilution water (resistivity > 18M¿).

Approximately 90% of the final volume of the test solution was added into test flasks. Five Daphnia magna aged from 6 to 24 hours were added into each test flask, which were then totally filled with the test solution (30 mL). Four replicates were prepared for each concentration. Four control flasks were prepared under the same conditions, with no test item.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS 7778-50-9)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

8.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 8.1-9.9

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 7.0-9.7

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Analytical results show that the test item concentrations strongly decreased during the exposition. As N,N DIETHYLHYDROXYLAMINE is known to be oxidized quickly in water by dissolved oxygen, it was decided to monitor the formation of the oxidation product in the test medium. Results obtained show that N,N DIETHYLHYDROXYLAMINE is quickly converted into a nitrone oxidation product in the conditions of the test. However, it was not possible to make a direct quantification of the nitrone product. Taking into account these information and that the test substance cannot be quantified at the end of the test at all concentrations (and thus at concentrations causing effects on daphnia), it was decided to use the initial measured concentrations for EC50 calculations.

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every month by performing an inhibition test on potassium dichromate. The nearest value of EC50 obtained on 19/09/12 was 0.75 mg/L.
For reference, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.

Reported statistics and error estimates:

The inhibition of mobility data are analyzed using an Excel program. It was designed to calculate the EC50 value and the 95% confidence interval. Probit analysis is generally used to calculate the 24 and 48-hours concentration, EC50 values. The No-Observed-Effect Concentration (NOEC) is determined by statistic analysis using the Dunnett’s test.

Dissolved O₂ and pH measured at the beginning and at the end (48h) of the exposition in the definitive test

Nominal concentration	pH		Dissolved O2(mg/L)
mg/L	T0	T48h	T0	T48h
0 (T)	8.03	7.79	8.6	8.7
1	8.04	7.84	8.6	8.6
2.2	8.05	7.87	8.4	8.3
4.8	8.02	7.92	8.2	8.0
10.6	8.05	8.04	7.7	8.0
23.4	8.06	8.17	6.9	6.7
51.4	8.12	8.41	5.9	5.0

At the highest concentrations tested, a decrease in dissolved O₂ concentration was observed from the beginning of the exposition. It may be related to the reaction of oxidation of N, with O₂. It has been considered that this depletion in oxygen may lead to the hypoxia of the medium and thus to the mobility of daphnia observed.ertheless, this hypothesis has been rejected since at concentrations causing effects on daphnia, concentration in dissolved O₂ was similar in the lowest test concentrations and in the control vessels.

Nominal and measured concentrations of the test item at the beginning and at the end of the exposure period in the definitive test

Concentrations of N,N DIETHYLHYDROXYLAMINE
Nominal Concentrations	Measured concentrations
	Initial	Final	Final/Initial
(mg/L)	(mg/L)	(mg/L)	%
0 (T)	< DL	< DL	-
1	1.2	< DL
2.2	2.5	< DL
4.8	5.8	< DL
10.6	11.55	< DL
23.4	29.95	< DL
51.4	69.60	< QL

< DL : concentration lower than the Detection Limit of the analytical method (0.008 mg/L)

< QL : concentration lower than the Quantification Limit of the analytical method (0.025 mg/L).

Definitive test - Percentage of Daphnia magna immobilisation

Nominal concentrations	Immobilisation (%)
(mg/L)	24 h	48 h
0 (T)	0	0
1	0	0
2.2	0	0
4.8	20	30
10.6	65	75
23.4	100	100
51.4	100	100

Validity criteria fulfilled:

yes

Remarks:

see details in conclusions

Conclusions:

According to these results, DEHA was observed to be toxic to aquatic invertebrates.
The study was performed in compliance with the following quality criteria:
- The immobilisation in the control did not exceed 10 per cent at the end of the test;
- Daphnids in the control were not trapped at the surface of the water;
- The dissolved oxygen concentration remained above 3 mg/L over the test period.

Executive summary:

Daphnia magna were exposed under static conditions to a range of concentrations of N,N DIETHYLHYDROXYLAMINE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of mobility over time periods of 24 and 48 hours. EC50_48h was measured to be 8.2 mg/L (based on initial measured concentrations). Validity criteria were fulfilled. Nevertheless, concentrations of DEHA were strongly decreased during the exposition. Indeed, DEHA is known to be oxidized quickly in water by dissolved oxygen. Thus EC50 values determined at 24 and 48h post exposure were based on initial measured concentrations.

Description of key information

 Daphnia magna were exposed under static conditions to a range of concentrations of N,N DIETHYLHYDROXYLAMINE dissolved in dilution water (Study report, 2012). The toxic effect measured during the assay was the inhibition of mobility over time periods of 24 and 48 hours. EC50_48h was measured to be 8.2 mg/L (based on initial measured concentrations). Validity criteria were fulfilled. Nevertheless, concentrations of DEHA were strongly decreased during the exposition. Indeed, DEHA is known to be oxidized quickly in water by dissolved oxygen. Thus EC50 values determined at 24 and 48h post exposure were based on initial measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.2 mg/L